The cloning of DNA segments is performed as a daily routine in many research labs. It is frequently performed in order to move a first polynucleotide sequence from a first vector into a second vector, where the second vector performs a function that is not performed by the first. Differences between the two vectors may include differences in selectable markers or differences in replicative sequences. They may also include differences in vector sequence elements that may directly interact with the first polynucleotide, for example by affecting expression of a gene encoded by the first polynucleotide, or by encoding polypeptides that interact with or are joined to polypeptides encoded by the first polynucleotide.
The specialized vectors used for subcloning DNA segments are functionally diverse. These include but are not limited to: vectors for expressing genes in various organisms; for regulating gene expression; for providing tags to modify polypeptide properties such as solubility, localization, affinity for a substrate, color, fluorescence, characteristics that facilitate protein purification and characteristics that facilitate tracking of proteins in cells; for modifying the cloned DNA segment (e.g., generating deletions); for the synthesis of probes (e.g., riboprobes); for the preparation of templates for DNA sequencing; for the identification of protein coding regions; for the fusion of various protein-coding regions; for expressing one or more enzymes to catalyze a reaction and for providing large amounts of the DNA of interest. It is common that a particular investigation will involve subcloning the DNA segment of interest into several different specialized vectors.
Simple subclonings, for example those where a single DNA segment to be subcloned is not large and the restriction sites are compatible with those of the subcloning vector, can be done in one day. However, it is frequently desirable to combine more than two polynucleotides, and the efficiency of such cloning reactions is significantly lower, resulting in more time, effort and money spent on creating the final desired DNA molecule. There is thus a need for a rapid and reliable method for moving multiple polynucleotides into one or more specialized vectors.